Fluorescence spectroscopy and microscopy for biology and medicine

Similar presentations

Presentation on theme: "Fluorescence spectroscopy and microscopy for biology and medicine"— Presentation transcript:

1 Fluorescence spectroscopy and microscopy for biology and medicineCZECH TECHNICAL UNIVERSITY IN PRAGUEFACULTY OF BIOMEDICAL ENGINEERINGFluorescence spectroscopy and microscopy for biology and medicineMartin Hof, Radek Macháň

2 Fluorescence spectroscopy and microscopy for biology and medicineCZECH TECHNICAL UNIVERSITY IN PRAGUEFACULTY OF BIOMEDICAL ENGINEERINGFluorescence spectroscopy and microscopy for biology and medicineMartin Hof, Radek MacháňAbsorption of light and electronic transitionsBasic principles of fluorescence, fluorescence spectraLifetime of fluorescence and its measurementQuenching of fluorescence and its biological applicationsAnisotropy of fluorescence and its biological applicationsInfluence of solvent on fluorescence spectraFoerster resonance energy transfer and excimer fluorescenceFluorescent proteinsFluorescence microscopy, confocal and 2-photon microscopyResolution of fluorescence microscope and its enhancementFluorescence correlation spectroscopyPhotodynamic Therapy

4 Also fluorescence is very, very, very sensitive! Why fluorescence?FluorescentProbeionselectric fieldsviscositypolaritypHtemperatureAlso fluorescence is very, very, very sensitive!Work with subnanomolar concentrations isroutine while femtomolarand even SINGLE MOLECULE studies arepossible with some effortit provides information on the molecular environmentit provides information on dynamic processes on the nanosecond timescaleFluorescence Probes are essentially molecular stopwatches which monitor dynamic events which occur during the excited state lifetime – such as movements of proteins or protein domains

7 A very brief history of the study of lightShowed that the component colors of the visible portion of white light can be separated through a prism, which acts to bend the light (refraction) in differing degrees according to the wavelength. Developed a “corpuscular” theory of light .1. Sir Isaac Newton 1672:2. Christian Huygens 1692:Developed a wave theory of light3. Hans Christian Oersted 1820Showed that there is a magnetic field associated with the flow of electric current4. Michael Faraday 1831Showed the converse i.e. that there is an electric current associated with a change of magnetic field

8 5. James Clark Maxwell: 1865Published his “Dynamical theory of the electromagnetic field” which combined the discoveries of Newton, Young, Foucault, Oersted and Faraday into a unified theory of electromagnetic radiationLight consists of electromagnetic transverse waves of frequency  and wavelength  related by  = nc where n is the index of refraction of the medium and c is the speed of the light in vacuum c = 3x1010 cm/sEBwe are interested in interactions of the electric field with the matter

12 Interaction of electromagnetic waves with matterAtoms and molecules described as electric multipoles, first approximation: electric dipoleClassical electrodynamics: dipoles oscillate at the frequency of the external electromagnetic field+-Elastic scattering of light

13 Interaction of molecules with photons - quantum descriptionLight exists in form of discrete quanta – photons E = hnAtoms and molecules occupy discrete energetic states, which can be found as the solution of Schroedinger’s equation.Exchange of energy with photons is accompanied by transitions between those states.rotational statesDJ =  1microwave regionvibrational statesDN =  1IR – VIS regionEelectronic statesUV – VIS region

14 Interaction of light with matter – overview of processeselastic scattering – no exchange of energy between the molecule and the photoninelastic (Raman) scattering – the photon either gives a part of its energy to the molecule or vice versaabsorption or emission of photons by the molecule12absorptionspontaneous emissioninduced emissioninduced emission is coherent with incident lightspontaneous emission by individual molecules is incoherentscattering is coherent and instantaneous

22 Electronic transitions from the ground state to the excited stateInter-nuclear distanceGS1v 0v 1v 2v 3v1 0v 11v 12v1 3Shaded areas reflects the probability of where the electron would be if it were in that vibrational bandMost favored transitions occur From themaximum shaded areas of the ground stateTo the maximum shaded areas of the excited state